Biologically active synthetic thyrotropin and cloned gene for producing same

ABSTRACT

Substantially pure recombinant TSH has been prepared from a clone comprising complete nucleotide sequence for the expression of the TSH. Diagnostic and therapeutic applications of the synthetic TSH are described.

[0001] The present invention is related generally to the isolation andcharacterization of new genes and proteins. More particularly, thepresent invention Is related to providing isolated, substantially pure,biologically active human thyrotropin (hTSH) synthesized by a clonedgene.

[0002] Thyrotropin (TSH) is a pituitary peptide hormone which regulatesimportant body functions. However, heretofore there was no stable,reliable and economic means of synthesizing this important hormone.

SUMMARY OF THE INVENTION

[0003] It is, therefore, an object of the present invention to providebiologically active, synthetic human thyrotropin in substantially pure,isolated form.

[0004] It is another object of the present invention to provide a clonedgene which directs the expression of biologically active humanthyrotropin in a suitable vector.

[0005] It is a further object of the present invention to provide anassay kit for measuring thyroid-stimulating hormone as well as otherthyrotropin substances such as thyroid-stimulating immunoglobulins andthe like.

[0006] It is a further object of the present invention to provide amethod of diagnosing and treating human thyroid cancer.

[0007] Other objects and advantages will become evident from thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] These and other objects, features and many of the attendantadvantages of the invention will be better understood upon a reading ofthe following detailed description when considered in connection withthe accompanying drawings wherein:

[0009]FIG. 1 shows schematic construction of the expression vectors.pSV2.G and pAV2 are pBR322 derived plasmids with the origin ofreplication (ori) and ampicillin resistance gene (amp r) as shown.pSV2.G contains the early promoter of SV40 upstream of the HindIIIcloning site, rabbit β-globin cDNA, and poly-adenylation site/intron ofSV40. pAV2 has the entire adenovirus-associated virus genome (4.7 kb)with its three promoters P5, P19, P40, and polyadenylation site. TheHindIII cloning site is downstream of the P40 promoter. Human TSHβ andhCGα was inserted into the HindIII site of either plasmid, formingpAV2-hTSHβ, pAV-hCGα, pSV2.GhTSHβ, and pSV2.G-hCGα.

[0010]FIG. 2 shows Northern blot analysis of transfected 293 and COScells. Total cellular RNA was separated on a 1% agarose-formaldehyde geland transferred to a nylon membrane. Forty micrograms of total RNA froma control of transfected cell culture were applied to each lane. HumanCGα and hTSHβ were abbreviated α and β in construct names and in otherfigures. Cells that were not transfected are labeled control. Cellstransfected with a calcium phosphate precipitate lacking DNA are labeledmock. Lanes 1-4 are total RNAs derived from 293 cells; lanes 5-9 areRNAs derived from COS cells. The migration position of an RNA standardin kilobases and hTSHβ mRNA from human pituitary is shown to the left ofthe autoradiograph. Below the autoradiograph is a simplified version ofFIG. 1 showing the pAV2 and pSV2.G plasmid as a single line, thepromoters as blackened circles, the 2.0 kb hTSHβ genomic fragment as abox containing two exons (blackened regions) and known polyadenylationsignal-site sequences as open arrowheads. Below each construct, pAV2-βand pSV2.G-β, is the predicted RNA initiating at the specified promoter,and splicing as shown. Solid arrowheads, poly(A) tails. Predicted sizein kilobases (kb) is shown to the right of each mRNA species.

[0011]FIG. 3 shows the results of gel chromatography. Cell medium from293 cells transfected with pAV2-hCGα/pAV2-hTSHβ/pVARNA waschromatographed on a Sephadex G-200 fine column. In addition, standardpreparations of hTSH, hCGα, and hTSHβ (described in the text) werechromatographed on the same column. RIA of human α and TSHβ and IRMA forhTSH was done on each 1.5-ml fraction. Elution position of bovinethyroglobulin (void, V_(o)), BSA [67,700 (67 k)] and ovalbumin [45,000(45 k)] is marked, as well as those of the standard preparations ofhTSH, hCGα, and hTSHβ.

[0012]FIG. 4 shows the results of human TSH IRMA. A highly sensitive andspecific hTSH IRMA was performed on two pituitary hTSH standards. WorldHealth Organization 80/558 (WHO STD) and NIH I-6 (I-6 STD), and themedium from 293 cells after transfection with pAV2-hCGα, pAV2-hTSHβ, andpVARNA (pAV2α/β/pVARNA). A logit transformation of assay binding wasplotted vs. arbitrary units of sample volume added to the assay.

[0013]FIG. 5 shows the results of in vitro bioassay of hTSH in ratthyroid cells. The human pituitary TSH standards and medium fromtransfected 293 cells used in this assay are defined in the legend toFIG. 4. This in vitro bioassay measures TSH stimulated ¹²⁵I uptake intorat thyroid cells (FRTL5). Iodide trapping by pituitary standards andmedium from a transfected culture are normalized to TSH immunoactivityin an IRMA.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The above and various other objects and advantages of the presentinvention are achieved by the cloning of complete nucleotide sequencewhich directs the synthesis of biologically active human thyrotropin ina suitable expression vector and isolating substantially pure form ofthe synthesized hormone.

[0015] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedhereunder are incorporated herein by reference. Unless mentionedotherwise, the techniques employed herein are standard methodologieswell known to one of ordinary skill in the art.

[0016] The term “substantially pure” as used herein means as pure as canbe obtained by employing standard conventional purification techniquesknown in the art.

[0017] The term “biologically active” as used herein means that therecombinant hormone, even though not identical in physical or chemicalstructure or composition as the naturally occurring hormone, yet isfunctionally equivalent thereto.

MATERIALS AND METHODS

[0018] Materials

[0019] Restriction and modifying enzymes were obtained from BethesdaResearch Laboratories (Gaithersburg, Md.) and Pharmacia (Piscataway,N.J.). ⁵²P and ⁵⁵S compounds were purchased from both DuPont New EnglandNuclear (Boston, Mass.) and Amersham/Searle Corporation (ArlingtonHeights, Ill.). Gene Screen and Gene Screen Plus membranes (New EnglandNuclear) were used in all DNA and RNA transfer procedures. Atransformation competent strain of Escherichia coli, HB101, was obtainedfrom Bethesda Research Laboratories and used in all transformations.Oligonucleotides were purchased from the Midland Certified ReagentCompany (Midland, Tex.). Cloning and propagation of DNA was done inaccordance with NIH guidelines. Sephadex G-200 fine and concanavalinA-Sepharose were obtained from Pharmacia Fine Chemicals. α-Methylglucoside and α-methyl mannoside were purchased from Sigma (St. Louis,Mo.). Human TSH, hCGα, and hTSHβ were provided by the NIDDK NationalHormone and Pituitary Program (Bethesda, Md.). Protein standards werepurchased from Sigma or Pierce Chemical Co. (Rockford, Ill.).

[0020] Genomic Screening

[0021] Independent recombinant phage clones (1×10⁵ ) of an EMBL3 humangenomic leukocyte library were screened for the presence of human TSHβusing a radiolabeled mouse TSHβ cDNA obtained from W. Chin, Brigham andWomen's Hospital, Harvard Medical School, Boston, Mass. and two separateclones were identified. A 34 base oligonucleotide, with the samesequence as the first 34 bases of the 5′-untranslated sequence of bovineTSHβ cDNA, was 5′-end labeled with [τ-₅₂P]ATP to a specific activity of5-8×10⁵ cpm/picomol using polynucleotide kinase; mouse TSHβ cDNA was[α-⁵²P ] dCTP labeled with a random primer to a specific activity of1-5×10⁵ cpm/μg. Both were used to probe Southern blots of restrictiondigests of one clone.

[0022] Subcloning and Sequencing

[0023] Genomic fragments were subcloned into pUC18 and mp13 tofacilitate restriction mapping and sequencing using the dideoxy chaintermination method of Sanger (Sanger et al, 1977, Proc Natl Acad Sci USA74:5463-5467).

[0024] Expression Vectors

[0025] A 621 bp hCGα cDNA (obtained from J. Fiddes, CaliforniaBiotechnology Inc., Palo Alto, Calif.) was inserted downstream of theearly promoter of SV40 in pSV2.G [obtained from B. Howard, NIH(Bethesda, Md.)] (Gorman et al, 1982, Mol Cell Biol 2:1044-1051) or theP40 promoter of adeno-associated virus in pAV2 (Laughlin et al, 1983,gene 23:65-73) at the Hind III site forming pSV2.G-hCGα and pAV2-hCGα(FIG. 1). HindIII linkers were ligated to a 2.0 kb PvuII fragment of thehTSHβ gene containing 277 bp of 5′-intron, both coding exons, a 450 bpintron, and approximately 800 bp of 3′-flanking DNA. It was insertedinto the same HindIII sites as hCGα forming pSV2.G-hTSHβ and pAV2-hTSHβ(FIG. 1). All plasmids were subjected to multiple restriction enzymedigestions to confirm the presence of only one insert in the properorientation.

[0026] Cell Culture

[0027] Adenovirus transformed human embryonal kidney cells (293 cells)and SV40 transformed monkey kidney cells (COS cells) were grown in amodified minimal essential (MEM) and Dulbecco's modified Eagle's medium,respectively. Both media were supplemented with 10% fetal bovine serum,4.4 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 250ng/ml amphotericin B. Twenty-four hours before transfection, the cellswere replated on 100-mm dishes at the same density (5×10 ⁵). On the dayof transfection fresh medium was added to the cells 4 h beforetransfection.

[0028] Transfection

[0029] All transfections were performed using the calcium phosphateprecipitation method (Graham et al, 1973, Virology 52:456-467). Theprecipitate was applied for 4 h, the cells were washed, and fresh mediumwas added. Total RNA was isolated according to the method of Cathala etal, 1983, DNA 2:329-335. The pAV2 plasmids were transfected into both293 and COS cells in Exp 1 and into only 293 cells in Exp 2. The pSV2.Gplasmids were only transfected into COS cells. When either the α- orβ-subunit was transfected alone into cells, 15 μg purified plasmid wereapplied to each plate. When both the α- and β-subunit werecotransfected, 9 μg each purified plasmid were applied to one plate. Insome cases, cells were cotransfected with pVARNA. pVARNA consists of anadenovirus type 2 DNA HindIII B fragment containing the genes for VA_(I)and VA_(II) inserted into the HindIII site of pBR322 (obtained fromKetner, Department of Biology, Johns Hopkins University, Baltimore,Md.). VA_(I) RNA stimulates translation by inhibiting phosphorylationand inactivation of the α subunit of eucaryotic initiation factor 2(Akusjarvi et al, 1987, Mol Cell Biol 7:549-551).

[0030] RNA Analysis, RIA, and IRMA

[0031] Northern blot analysis of total RNA from transfections wasperformed using standard methods (Maniatis et al, 1982, MolecularCloning, ed 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,p 202) and manufacturer's specifications. Common human α-subunit RIA,hTSHβ-subunit RIA, and hTSH IRMA were performed in duplicate on themedium from each transfected culture (McBride et al, 1985, Clin Chem31:1865-1867; Kourides et al, 1974, Endocrinology 94:1411-1421). Thesensitivities of the assays were less than 0.03 ng/ml, less than 0.03ng/ml, and less than 0.06 ng/ml, respectively. Cross-reactivity betweenthe corresponding subunit and hTSH, at the measured concentration, wasless than 5% in the common α and less than 2% in the hTSHβ RIA (Kourideset al, supra). In addition, at the measured free subunit concentrations,the hTSH IRMA exhibited less than 1% cross-reactivity (data not shown).

[0032] Gel and Lectin Affinity Chromatography

[0033] The apparent mol wt of hTSH synthesized in 293 cells wasdetermined by gel chromatography on a 1.5×90-cm Sephadex G-200 finecolumn calibrated during each chromatography run with five proteinstandards (bovine thyroglobulin, BSA, ovalbumin, bovine chymotrypsinogenA, and whale myoglobin). The column was equilibrated and run at 4° C. ina buffer containing 0.12 M sodium chloride. 0.1 M borate, and 0.02%(wt/vol) sodium azide, pH 7.4. Two milliliters of fresh MEM mediumcontaining 100 μUhTSH (WHO80/558), 100 ng hCGα (CR-119), and 100 nghTSHβ (AFP-3929β) were applied during chromatography of standardpreparations. The column was washed and then 2 ml MEM medium from 293cells transfected with pAV2-hCGα/pAV2-hTSHβ/pVARNA were applied.Fractions of 1.5 ml were collected at a flow rate of 6 ml/h.

[0034] The binding of hTSH synthesized in 293 cells to concanavalinA-Sepharose was also determined using methods previously described(Gesundheit et al, 1987, J Biol Chem 262:5197-5203). Samples wereapplied to lectin column, and 2-ml fractions were collected at a flowrate of 10 ml/h. Human TSH IRMA was common human α- and hTSHβ-subunitRIAs were performed on fractions from gel and lectin chromatography.Recovery of hTSH and its free subunits was generally greater than 90%from chromatography.

[0035] TSH Bioassay

[0036] Thyrotropic bioactivity was measured as the ability to stimulatethe uptake of ¹²⁵I into rat thyroid cells (PRTL5) in accordance with theprocedure of Dahlberg et al, 1987, J Clin Invest 79:1388-1394. Thisassay measures human, rat, and bovine thyrotropin but not gonadotropinsor free α- or TSHβ-subunits. Sample determinations were performed induplicate and compared to two pituitary hTSH standards (World HealthOrganization 80/558 and National Institutes of Health I-6). Results areexpressed as microunits per ml; one microunit of WHO 80/558 isequivalent to 0.09 ng of NIH I-6 purified hTSH (unpublished data).

[0037] Statistics

[0038] Significant differences in immunoassay of cell media from variouscontrol and transfected cultures were determined using Student's t test.

RESULTS

[0039] Human TSHβ Gene

[0040] A 17 kb genomic fragment was isolated by screening 1×10⁵recombinant phage clones. A restriction map (FIG. 1) was constructedusing Southern blots of phage DNA hybridized with both a mouse TSHβ cDNAprobe (lacking 5′-untranslated sequences) and a 34 bp bovine5′-untranslated sequence probe. Two coding exons are separated by a 450bp intron and the sequence is identical to the published partialsequence (Hayashizaki et al, 1985, FEBS Lett 188:394-400) (data notshown). However, the complete coding sequence was not heretofore known.

[0041] Transfection

[0042] Two experiments were performed to compare the level of mRNA andprotein production between the most active adeno-associated viruspromoter, P40 and the early promoter of SV40.

[0043] Table 1 shows the RIA and IRMA assay results from these twoexperiments. Interestingly, the 293 cells synthesized small amounts offree α-subunit (control) whose levels were increased approximately10-fold in a transfection with the calcium phosphate precipitate butwithout DNA (mock) (P<0.0005). While neither transfection with pAV2-hCGαnor pVARNA increased α-production above the level of a mocktransfection, the combination increased free α-levels 3- to 5-fold(P<0.0005). Thus exogenous sources of the human α-subunit were clearlyimportant in mediating this increase. The same pattern of pVARNAincreasing protein production was seen when pAV2-hTSHβ was transfected.293 cells do not produce hTSHβ so that the medium of cells exposed to amock or pVARNA transfection did not have measurable hTSHβ. Only the 293cells exposed to pAV2-hTSHβ produced hTSH. When the β plasmid wastransfected alone the hTSH formed was due to combination with endogenousα. Cotransfection with both α- and β-plasmids, though, increased hTSHlevels 1.5- to 2-fold (P<0.05).

[0044] COS cells synthesized neither free α nor β but could synthesizehTSHβ and hTSH when transfected with the appropriate plasmids. Thelevels of protein production were 10- to 100-fold less than in 293 cellsand were only measurable when the pVARNA plasmid was also transfected.Regardless of whether pAV2 or PSV2.G was used, protein levels werebarely if at all measurable without the pVARNA plasmid.

[0045]FIG. 2 shows a Northern blot of total cellular RNA hybridized withthe mouse TSHβ cDNA probe. In 293 cells, hTSHβ message was not detectedfrom nontransfected (control) or from mock transfected cells. However,three RNA species of 2.3 kb, 1.6 kb, and 650 bases were noted aftertransfection of pAV2-hTSHβ and pVARNA (lane 3). These three bands arethe same size as those predicted from pAV2-hTSHβ if transcription beganat all three adeno-associated viral promoters (FIG. 2). The mRNA of 650bases presumably represent a properly spliced hTSHβ message. Lane 4shows the same three bands but at lesser intensity when pAV2-hCGα,pAV2-hTSHβ, and pVARNA were cotransfected. This reduction in signalintensity seen in lane 4 may have been due to the reduction in theamount of pAV2-hTSHβ transfected from 15 μto 9 μg.

[0046] Control and mock transfected COS cells also did not contain hTSHβmessage. When pSV2.G-hTSHβ was transfected (lanes 7-9), a major band of900 bases and a minor band of 3.0 kb were seen. Without being bound toany theory, it is postulated that the 900 base species could represent amRNA with the 277 bases of 5′-intron remaining, while the 3.0 kb speciescould represent read through of the hTSHβ polyadenylation signal-siteand use of the polyadenylation signal-site of pSV2.G (See FIG. 2).

[0047] Specific human α mRNA transcripts of appropriate size analogousto the hTSHβ mRNA above were observed in cells transfected withpAV2-hCGα (data not shown). Since the main object of this invention isprotein expression, the relative contribution of human α mRNA fromendogenous vs. exogenous (pAV2-hCGα) sources in 293 cells was notdetermined. However, the data suggest that the high level of freeα-subunit observed after transfection with pAV2-hCGα/pVARNA is mostlikely due to mRNA from exogenous sources.

[0048] Gel and Lectin Affinity Chromatography

[0049] The apparent molecular weight of hTSH and its subunitssynthesized in 293 cells after transfection withpAV2-hCGα/pAV2-hTSHβ/pVARNA was determined on a G-200 Sephadex column(FIG. 3). In addition, standard preparations of hTSH, hCGα, and hTSHβwere chromatographed on the same column. Internal protein standards hadidentical elution patterns between runs as determined by optical densityat 280 nm. In each case, the apparent mol wt of synthetic hTSH and itssubunits was larger than its corresponding standard. Specifically,synthetic hTSH displayed an apparent mol wt of 45,000 and was largerthan standard pituitary hTSH (apparent M_(r)=40,000). This clearlyindicates that the recombinant TSH is not constitutively identical tothe natural product. The human α and hTSHβ from transfection coelutedwith the hTSH pituitary standard, and both were larger than theirrespective standard subunit preparation. However, in the case of freehuman α-subunit, the relative contribution to this chromatographypattern of endogenous α as compared to exogenous α from pAV2-hCGα cannotbe determined.

[0050] The binding pattern to concanavalin A-Sepharose of synthetic hTSHfrom 293 cells as compared to standard human pituitary hTSH is shown inTable 2. Synthetic hTSH was glycosylated as indicated by completebinding to concanavalin A-Sepharose. The different elution pattern ofstandard vs. synthetic hTSH from the lectin columns is indicative of adifference at least in carbohydrate structure, again showing that therecombinant TSH (rTSH) is distinctly different from the naturallyoccurring TSH.

[0051] Immunoactivity and Bioactivity

[0052]FIG. 4 shows that the hTSH produced in cell culture wasindistinguishable from two pituitary hTSH standards in an assayinvolving two antibodies directed at different epitopes of the hTSHheterodimer (McBride et al, supra). The slopes were parallel over theentire range of values. FIG. 5 shows the same hTSH in a ¹²⁵I trapping invitro TSH bioassay compared to the same pituitary hTSH standards. The invitro bioassay of standard pituitary hTSH or the cell culture productfrom 293 cells (pAV2-hCGα/pAV2-hTSHβ/pVARNA) was normalized toimmunoreactivity in a hTSH immunoradiometric assay (IRMA) assay. Thedose response and ED₅₀ of the standards and cell culture product wereidentical. In addition, the cell culture product from COS cells(pAV2-hCGα/pSV2β/pVARNA) was biologically active although the lowerlevel of expression prevented determination of a dose response curve.

[0053] In summary, a 17 kb genomic fragment of hTSHβ has been isolatedand both coding exons of this gene produced hTSHβ and hTSH in atransient expression assay. This is the first report of TSH from anyspecies produced by gene transfection in cell culture. The expressionvectors of hTSHβ included only the two coding exons, and not the5′-untranslated exon of the gene (Wondisford et al, Mol. Endo. 2(1):32-39, 1988).

[0054] Transient expression after gene transfection was used to testboth the early promoter of SV40 or the P40 promoter of adeno-associatedvirus. The early promoter in COS cells produced more mRNA than the P40promoter in 293 cells regardless of whether pVARNA was cotransfected.However, pVARNA clearly increased mRNA levels in either vector system.This suggests that in addition to increasing the rate of translation,pVARNA must either increase transcriptional rate, RNA transport, orstability.

[0055] While the pSV2.G-hTSHβ expression vector produced higher levelsof hTSHβ mRNA than pAV2-hTSHβ, this mRNA was about 250 bases larger thanthat found in the human pituitary. The 450 bp intron was certainlyspliced out since this intron in the mature message would have preventedhTSHβ protein synthesis. Also, an mRNA of appropriate size was producedby pAV2-hTSHβ indicating that the polyadenylation site in the fragmentmust be active. Thus, the most likely reason for a larger hTSHβ mRNA inCOS cells was the lack of splicing of a 277 bp intron fragment upstreamof the first coding exon. Eighteen base pairs downstream of thetranscriptional start site of the P40 promoter is a consensus splicedonor site which could explain why the 277 bp intron fragment would bespliced out in the adeno-associated virus vector.

[0056] The plasmid, pVARNA, increased protein production in eithervector system, but the P40 promoter in 293 cells led to expression ofbetween 10- to 100-fold more protein than the early promoter of SV40when cotransfected with pVARNA. This is most likely due to an increasedtranslational rate mediated by pVARNA as has been previouslydemonstrated for expression of other mRNAs (Akusjarvi et al, 1987, MolCell Biol 7:549-551). Of course, the possibility that the larger mRNAfrom pSV2.G-hTSHβ contributed to the lower protein levels from COS cellscannot be excluded

[0057] The hTSH produced in cell culture was functionallyindistinguishable from two pituitary hTSH standards in both a highlyspecific IRMA and in vitro bioassay. It should be noted, however, thatthe synthetic hTSH of the present invention was larger in size thanstandard pituitary hTSH on gel chromatography. Although it wasglycosylated as indicated by complete binding to concanavalin A, itdisplayed a somewhat different pattern on lectin chromatography ascompared to a standard hTSH preparation. The larger mol wt of thesesynthetic glycoproteins as compared to pituitary standards is mostlikely due to an altered glycosylation pattern such as more sialylation.In the case of hTSH, this might also reflect a β-subunit containing the118 amino acids predicted from the nucleic acid sequence rather than the112 found in standard hTSH purified from postmortem human pituitaries.

[0058] Transient expression is more convenient than stable integrationin the analysis of a large number of expression vectors. pAV2 and pVARNAnow allow transient expression of hTSH in 293 cells at levels highenough to analyze protein and glycosylation site structure-functionrelationships. Previously, the only information about such relationshipscame from studies involving chemical modifications of protein byiodination, nitration, acetylation, and carboxymethylation (Pierce etal, 1981, Annu Rev. Biochem., Annual Reviews Inc., Palo Alto, Calif., pp465-495) or inhibition of glycosylation by tunicamycin (Weintraub, etal, 1980, J Biol Chem 255:5715-5723). The chemical groups couldthemselves change protein conformation irrespective of the alteration inamino acids they produce and inhibition of glycosylation affects notonly TSH but all cellular glycoproteins. Site-directed mutagenesis ofthe hCGα cDNA and human TSHβ-gene could directly address what regionsare important for protein conformation, subunit combination, receptorbinding, biological activity, and metabolic clearance withoutintroducing chemical groups or unknown changes into the proteinstructure.

[0059] The availability of substantially pure rTSH now makes thediagnosis and treatment of human thyroid cancer and the determination ofthe level of TSH a reality.

[0060] Currently, the only available method to diagnose and treat humanthyroid cancer involves making patients hypothyroid and allowing theirown endogenous human TSH to rise after several weeks to stimulate theuptake of ¹³¹I into the cancer. Such stimulation is used as a diagnostictest to localize the tumor by scanning and is subsequently used to treatthe cancer by giving large doses of ¹⁵²I. All of the diagnostic testsand therapies depend on high levels of human TSH. However, the techniqueof producing endogenous hypothyroidism has disabling side effectsincluding lethargy, weakness, cardiac failure, and may also lead to arapid growth of the tumor over the several week period of treatment. Incontrast, if a desirable form of synthetic human TSH were available,patients could be treated while they were euthyroid by giving exogenousinjection of the TSH. However, presently it is not feasible to giveexogenous TSH because there is not enough natural product from availablehuman pituitaries collected at autopsies. Furthermore, even ifavailable, the human pituitaries have been found to be contaminated withviruses and the National Pituitary Agency has forbidden the use of thenatural product for any human diagnostic or therapeutic studies. This istrue for all human pituitary hormones including human growth hormonewhich is now exclusively marketed as a synthetic product. However, thetechnology that was applicable for human growth hormone (anon-glycoprotein) is not at all applicable for human TSH (a glycoproteinhormone of two glycosylated subunits). As has been described hereinsupra, only the methodology described herein relating to transfectionand proper glycosylation of each subunit in mammalian cells produces adesirable biologically active rTSH material. Moreover, it has been foundthat the altered glycosylation pattern that can be achieved with thecells, as described in the methodology of the present invention,produces a longer acting human thyrotropin which is particularly suitedfor the diagnosis and treatment of thyroid cancer.

[0061] The diagnosis and treatment of thyroid cancer involves firstpurifying the synthetic TSH from large volumes of tissue culture mediaharvested from approximately ten billion cells over two to four weeks.Using a chemically defined medium to reduce protein contaminants as iswell known in the art, synthetic human TSH, which represents about fiveto ten percent of all the protein secreted into the medium, can beobtained. The human TSH thus obtained is then purified by a combinationof standard techniques including immunoaffinity chromatography, HPLCexclusion chromatography (repeated two to three times) followed bydialysis and concentration by ultrafiltration, lyophilization and thelike. The purified human TSH is then tested in animals to assure itsefficacy as well as freedom from any unexpected toxicity. The syntheticTSH is then tested in patients in clinical trials using different dosesto determine the optimal doses to achieve maximal uptake into the tumorfor both diagnosis and treatment with ¹³¹I. During initial try-outs fordiagnosis, one to two administrations of about 100 μg, while duringtherapy three to six doses of about 100 to 200 μg may be administered,but the optimal dose schedule is determined by the results of theclinical trials. It is noted that all of these procedures areaccomplished while the patient is still euthyroid without producing anyof the disabling side effects of hypothyroidism which are otherwiseencountered in the methods heretofore available.

[0062] When the optimal uptake of ¹³¹I has been established, patientsmay be treated with doses of about 50 to 400 mCi of ¹³¹I and the effectof therapy assessed by subsequent ¹³¹I diagnostic tests as well asconventional x-rays, CAT scans, measurement of serum thyroglobulin andthe like. Of course, ¹³¹I-labeled rTSH, which is produced by standardmethodology well known in the art, can be appropriately utilized in theprocedures mentioned above.

[0063] It is estimated that there will occur about ten thousand newcases of thyroid cancer in the United States each year and a very largeprevalence of older cases of this cancer require repeated diagnostic andtherapeutic intervention which are currently unsatisfactory.Availability of synthetic human TSH as taught herein, even at a cost of$50.00 to $100.00 per injection, will still be a relatively inexpensivepart of the complete evaluation and therapy for this difficult, butcurable cancer.

[0064] Another advantage of the product of the present invention is toprovide assay components for human thyrotropin using the technique ofradioimmunoassay. Certain immunoassay kits are presently available, butthe reagents therein are again derived from a very short supply ofnatural product. Moreover, the natural product varies greatly dependingon the source of the human pituitaries as well as the degree ofdegradation that occurs during autopsy. This has led to considerablevariation among commercially available kits with disagreements inresults of the TSH testing among various kits. In contrast, the presentinvention, for the first time, provides a virtually unlimited supply ofa stable preparation of synthetic TSH allowing kit manufacturers to havea universal standard preparation and a virtually identical andinexhaustible supply of the reagents. This would allow world wideconsistency of dosage and lead to much needed standardization in themeasurement of human TSH which is vital in the assessment of thyroidfunction in humans. This is accomplished by labeling the rTSH withradioactive iodine (I¹³¹, I¹²⁵)or another suitable labeling materialsuch as chemoluminiscent or fluorescent labels and by producingantibodies to the pure product by either polyclonal or monoclonaltechniques which are well known in the art and providing inexhaustiblesupplies of immunoglobulins without significant interferingcross-reactivity with other hormones. The antibodies are then formulatedin classic radioimmunoassay kits which are supplied to the manufacturersto be used in a variety of standard assay methodologies (RIA, IRMA,Sandwich Assays and the like).

[0065] There are various other advantages of rTSH. Tests havedemonstrated that it is possible to modify the TSH by expressing thehormone in various cell lines leading to altered glycosylation patterns.Moreover, using the technique of site-directed mutagenesis wherebyindividual bases in the DNA are changed, products are obtained withaltered biologic function such as prolonged or decreased half life, aswell as competitive antagonists that bind to the TSH receptor andactually block TSH function. Such competitive antagonists are useful ina novel way to treat diseases such as TSH-induced byperthyroidism aswell as Graves' disease which is caused by auto-antibodies to the TSHreceptor. The function of these abnormal stimulators would be blocked bythe competitive antagonist that we have already shown to be active atthe cellular level. Moreover, using various long-acting and short-actingpreparations, superagonists can be prepared which would be particularlystimulating to thyroid function, and superantagonists can be preparedwhich would be particularly inhibitory of thyroid function. In thismanner, thyroid function can be controlled in many different types ofdisease of thyroid overactivity or underactivity. It should be notedthat these completely novel approaches are feasible only because of theavailability of the synthetic TSH by the methodology of the presentinvention because, for the reasons mentioned above, the natural productis prohibited from such in vivo use.

[0066] It has also been discovered that modifications of thetransfection process greatly enhances the amount of TSH production bymammalian cells. For example, instead of using TSH-β gene constructscontaining only the 2nd and 3rd exons (the 2 coding exons), a newconstruct is made by adding the first untranslated exon of TSH-β. Theinclusion of this untranslated TSH-β exon greatly increases TSHproduction. Without being bound to any specific theory, it is postulatedthat the enhanced TSH production occurs by increased transcription rateand/or mRNA stability. Moreover, it has been discovered that an excessof the α gene in a ratio of about 3 to 5 times greater than the β gene,yields high rate of TSH production (10-50 mg/month), close to commercialscale production.

[0067] A standard concentration curve utilizing anti-rTSH antibodies isestablished to determine the amount of TSH in a sample by conventionalimmunological assays.

[0068] In summary, a recombinantly made synthetic TSH has been madewhich, even though constitutively distinct from the natural product,possesses functional properties similar to the natural product and isuseful for diagnostic as well as therapeutic purposes.

[0069] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. TABLE 1 Immunoassay forHuman TSH and its Subunits in Cell Media from Control and TransfectedCultures Human Human TSH Transfected Construct Human TSHβ IRMA orControl αRIA (ng/ml) RIA (ng/ml) (μU/ml) (Exp no.) n Mean SEM Mean SEMMean SEM 293 cells Medium (1,2) <0.03 <0.03 <0.6 Control (2) 4 0.310.01^(a) <0.03 <0.6 Mock (2) 4 3.4 0.36^(b) <0.03 <0.6 pVARNA(2) 4 5.00.68  <0.03 <0.6 pAV2(2) 4 2.1 0.11  <0.03 <0.6 pAV2α(2) 4 1.6 0.31 <0.03 <0.6 pAV2α/pVARNA(2) 4 17.3 1.0  <0.03 <0.6 pAV2β(2) 4 3.4 0.37 <0.03 <0.6 pAV2β/pVARNA(1) 2 2.2 0.30  1.5 0.09  2.8 0.30  (2) 4 5.70.64  3.3 0.33  6.9 0.64^(c) pAV2α/β/pVARNA(1) 2 4.0 1.5  0.6 0.24  11.26.3  (2) 4 6.1 0.54  1.1 0.09^(d) 15.1 3.6  COS Cells Medium (1,2) <0.03<0.03 <0.6 Control (2) 2 <0.03 <0.03 <0.6 Mock (2) 2 <0.03 <0.03 <0.6pAV2α/pVARNA(1) 2 0.06 0^(b  ) <0.03 <0.6 pSV2β(1) 2 <0.03 0.04 0^(d  )<0.6 (2) 2 <0.03 <0.03 <0.6 pSV2β/pVARNA(2) 2 <0.03 0.16 0.01^(d) <0.6pSV2α/β(2) 2 <0.03 <0.03 <0.6 pAV2α/pSV2β/ 2 0.09 0.02^(b) 0.10 0.02^(d)0.9 0.06^(c) pVARNA(1) # cells. Control, Medium from nontransfectedcells. Mock, Medium from cells transfected with a calcium phosphateprecipitate lacking DNA.

[0070] TABLE 2 Lectin Chromatography of Synthetic and Standard hTSHBound- Unbound MG Column (%) (%) Bound-MM 1 WHO STD 0 12 88 2pAV2α/β/pVARNA 0 28 72

What is claimed is:
 1. Substantially pure, biologically activerecombinant human thyrotropin (rTSH).
 2. The thyrotropin of claim 1labeled isotopically with I¹³¹, I¹²⁵, chemoluminiscently orfluoroscently.
 3. The thyrotropin of claim 1 produced by recombinantgenetic process using constructs with gene elements that enhancethyrotropin production.
 4. A clone comprising complete nucleotidesequence for the expression of the thyrotropin of claim 1 in a suitableexpression vector.
 5. The clone of claim 4 further comprising firstuntranslated exon of TSH-β.
 6. A method for producing TSH, comprising(a) allowing expression of TSH by the clone of claim 4 in a suitableexpression vector; and (b) then recovering substantially pure TSH byconventional purification and isolation methodology.
 7. A method forproducing TSH, comprising (a) allowing expression of TSH by the clone ofclaim 5 in a suitable expression vector; and (b) then recoveringsubstantially pure TSH by conventional purification and isolationmethodology.
 8. A method for producing TSH, comprising (a) allowingexpression of TSH by the clone of claim 4 , wherein TSHα is about 3 to 5times in excess of TSHβ; (b) then recovering substantially pure TSH byconventional purification and isolation methodology.
 9. A method forproducing TSH, comprising (a) allowing expression of TSH by the clone ofclaim 5 , wherein TSHα is about 3 to 5 times in excess of TSHβ; (b) thenrecovering substantially pure TSH by conventional purification andisolation methodology.
 10. A TSH antagonist produced by a mutant of theclone of claim 4 .
 11. A TSH agonist produced by a mutant of the cloneof claim 4 .
 12. A kit comprising containers separately containing (a)universal standard of substantially pure unlabeled rTSH; (b)substantially pure, labeled rTSH; (c) antibodies against purified rTSH;and (d) instructional material describing the use of reagents (a), (b)and (c).
 13. Anti-rTSH antibodies without interfering cross-reactivitywith non-TSH hormones.
 14. A method for determining the level of TSH ina sample comprising reacting an aliquot of a sample in which the amountof TSH is to be determined with the antibodies of claim 13 and comparingthe level of antibody reactivity with a predetermined standardantibody-rTSH reactivity curve to determine the amount of TSH present insaid sample.
 15. A method of diagnosing the extent of thyroid cancer,comprising administering rTSH of claim 1 to a patient to maximize ¹³¹Iuptake, and then administering a visualizing dose of ¹³¹I to saidpatient; and then visualizing the cancer by standard visualizing means.16. A method for treating thyroid cancer, comprising administeringtherapeutic regimen of a combination of rTSH of claim 1 and ¹³¹I or ¹³¹Ilabeled rTSH to a patient afflicted with thyroid cancer.
 17. A method ofblocking TSH activity, comprising inhibiting TSH activity by competitiveamount of the antagonist of claim 10 .
 18. A method of stimulating TSHactivity, comprising inducing TSH production by the agonist of claim 11.